This invention relates to systems and methods for chemical analysis, and in particular to a novel reaction vessel and temperature control system.
There are many applications in the field of chemical processing in which it is desirable to precisely control the temperature of a biological sample, to induce rapid temperature changes in the sample, and to detect target analytes in the sample. Applications for such heat-exchanging chemical reactions may encompass organic, inorganic, biochemical or molecular reactions. Examples of thermal chemical reactions include isothermal nucleic acid amplification, thermal cycling nucleic acid amplification, such as the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication, enzyme kinetic studies, homogeneous ligand binding assays, and more complex biochemical mechanistic studies that require complex temperature changes. Temperature control systems also enable the study of certain physiologic processes where a constant and accurate temperature is required.
One of the most popular uses of temperature control systems is for the performance of PCR to amplify a segment of nucleic acid. In this well known methodology, a DNA template is used with a thermostable DNA polymerase, nucleoside triphosphates, and two oligonucleotides with different sequences, complementary to sequences that lie on opposite strands of the template DNA and which flank the segment of DNA that is to be amplified (xe2x80x9cprimersxe2x80x9d). The reaction components are cycled between a higher temperature (e.g., 95xc2x0 C.) for dehybridizing double stranded template DNA, followed by lower temperatures (e.g., 40-60xc2x0 C. for annealing of primers and 70-75xc2x0 C. for polymerization). Repeated cycling between dehybridization, annealing, and polymerization temperatures provides exponential amplification of the template DNA.
Nucleic acid amplification may be applied to the diagnosis of genetic disorders; the detection of nucleic acid sequences of pathogenic organisms in a variety of samples including blood, tissue, environmental, air borne, and the like; the genetic identification of a variety of samples including forensic, agricultural, veterinarian, and the like; the analysis of mutations in activated oncogenes, detection of contaminants in samples such as food; and in many other aspects of molecular biology. Polynucleotide amplification assays can be used in a wide range of applications such as the generation of specific sequences of cloned double-stranded DNA for use as probes, the generation of probes specific for uncloned genes by selective amplification of particular segments of cDNA, the generation of libraries of cDNA from small amounts of mRNA, the generation of large amounts of DNA for sequencing and the analysis of mutations.
A preferred detection technique for chemical or biochemical analysis is optical interrogation, typically using fluorescence or chemiluminescence measurements. For ligand-binding assays, time-resolved fluorescence, fluorescence polarization, or optical absorption is often used. For PCR assays, fluorescence chemistries are often employed.
Conventional instruments for conducting thermal reactions and for optically detecting the reaction products typically incorporate a block of metal having as many as ninety-six conical reaction tubes. The metal block is heated and cooled either by a Peltier heating/cooling apparatus or by a closed-loop liquid heating/cooling system in which liquid flows through channels machined into the block. Such instruments incorporating a metal block are described in U.S. Pat. No. 5,038,852 to Johnson and U.S. Pat. No. 5,333,675 to Mullis.
These conventional instruments have several disadvantages. First, due to the large thermal mass of a metal block, the heating and cooling rates in these instruments are limited to about 1xc2x0 C./sec resulting in longer processing times. For example, in a typical PCR application, fifty cycles may require two or more hours to complete. With these relatively slow heating and cooling rates, some processes requiring precise temperature control are inefficient. For example, reactions may occur at the intermediate temperatures, creating unwanted and interfering side products, such as PCR xe2x80x9cprimer-dimersxe2x80x9d or anomalous amplicons, which are detrimental to the analytical process. Poor control of temperature also results in over-consumption of expensive reagents necessary for the intended reaction.
A second disadvantage of these conventional instruments is that they typically do not permit real-time optical detection or continuous optical monitoring of the chemical reaction. For example, in conventional thermal cycling instruments optical fluorescence detection is typically accomplished by guiding an optical fiber to each of ninety-six reaction sites in a metal block. A central high power laser sequentially excites each reaction site and captures the fluorescence signal through the optical fiber. Since all of the reaction sites are sequentially excited by a single laser and since the fluorescence is detected by a single spectrometer and photomultiplier tube, simultaneous monitoring of each reaction site is not possible.
Some of the instrumentation for newer processes requiring faster thermal cycling times has recently become available. One such device is disclosed by Northrup et al. in U.S. Pat. No. 5,589,136. The device includes a silicon-based, sleeve-type reaction chamber that combines heaters, such as doped polysilicon for heating, and bulk silicon for convection cooling. The device optionally includes a secondary tube (e.g., plastic) for holding the sample. In operation, the tube containing the sample is inserted into the silicon sleeve. Each sleeve also has its own associated optical excitation source and fluorescence detector for obtaining real-time optical data. This device permits faster heating and cooling rates than the instruments incorporating a metal block described above. There are, however, several disadvantages to this device in its use of a micromachined silicon sleeve. A first disadvantage is that the brittle silicon sleeve may crack and chip. A second disadvantage is that it is difficult to micromachine the silicon sleeve with sufficient accuracy and precision to allow the sleeve to precisely accept a plastic tube that holds the sample. Consequently, the plastic tube may not establish optimal thermal contact with the silicon sleeve.
The present invention overcomes the disadvantages of the prior art by providing an improved instrument and reaction vessel for thermally controlling and optically interrogating a sample. In contrast to the prior art instruments described above, the system of the present invention permits extremely rapid heating and cooling of the sample, ensures optimal thermal transfer between the sample and heating or cooling elements, and provides for real-time optical detection and monitoring of the sample with increased detection sensitivity.
In a preferred embodiment, the system of the present invention includes a reaction vessel for holding a sample for chemical reaction and optical detection. The vessel has a rigid frame defining the side walls of a reaction chamber, and at least one flexible sheet attached to the rigid frame to form a major wall of the chamber. The vessel also includes a loading structure extending from the frame for loading a sample into the chamber. The loading structure defines a loading reservoir in fluid communication with the chamber. The loading reservoir receives the sample prior to loading the sample into the chamber. The loading structure also includes an aspiration port in fluid communication with the chamber.
The system also includes an aspiration and dispensing device, such as a pipette or syringe, for dispensing the sample into the loading reservoir, for subsequently establishing a seal with the aspiration port, and for drawing the sample from the loading reservoir into the chamber by vacuum. Loading the sample into the chamber in this manner reduces the likelihood that air bubbles form in the chamber during the sample loading process. Air bubbles would significantly harm subsequent optical detection of target analytes in the sample. The loading reservoir and aspiration port also eliminate the need to insert the pipette into the chamber. Consequently, the thickness of the chamber is not limited by the minimum practical pipette diameter, which can be employed in the sample transfer process.
The system also includes at least one thermal surface for contacting the flexible major wall of the chamber. The system further includes a device for increasing the pressure in the chamber. The pressure increase in the chamber is sufficient to force the flexible major wall to contact and conform to the thermal surface, thus ensuring optimal thermal conductance between the thermal surface and the chamber. The system also includes one or more thermal elements (e.g., a heating element, thermoelectric device, heat sink, fan, or Peltier device) for heating or cooling the thermal surface to induce a temperature change within the chamber.
In the preferred embodiment, the reaction vessel includes first and second flexible sheets attached to opposite sides of the rigid frame to form opposing major walls of the chamber. In this embodiment, the system includes first and second thermal surfaces formed by first and second opposing plates positioned to receive the chamber of the vessel between. When the pressure in the chamber is increased, the flexible major-walls expand outwardly to contact and conform to the inner surfaces of the plates. A resistive heating element, such as a thick or thin film resistor, is coupled to each plate for heating the plates. In addition, the system includes a cooling device, such as a fan, for cooling the plates. Each of the plates is preferably constructed of a ceramic material and has a thickness less than or equal to 1 mm for low thermal mass. In particular, it is presently preferred that each of the plates have a thermal mass less than about 5 J/xc2x0 C., more preferably less than 3 J/xc2x0 C., and most preferably less than 1 J/xc2x0 C. to enable extremely rapid heating and cooling rates.
The pressurization of the chamber ensures that the flexible major walls of the vessel contact and conform to the inner surfaces of the plates, thus guaranteeing optimal thermal conductance between the major walls and the plates. In the preferred embodiment, the vessel includes a seal aperture extending over an outer end of the loading reservoir and an outer end of the aspiration port, and the device for pressurizing the chamber comprises a plug which is inserted into the aperture to seal the aperture and to compress gas in the vessel, thereby increasing pressure in the chamber. The plug also simultaneously seals the chamber, loading reservoir, and aspiration port from the environment external to the vessel. The reaction vessel may be filled and pressurized manually by a human operator, or alternatively, the system may include an automated machine for filling and pressurizing the vessel. In this automated embodiment, the system preferably includes a pipette for filling the vessel and a pick-and-place machine for inserting the plug into the seal aperture after filling. The plug preferably includes a cap having a tapered engagement aperture for receiving and establishing a fit with a machine tip, thereby enabling the machine tip to pick and place the plug into the aperture. optionally, the cap may include latches extending from its sides, and the vessel may include catches for engaging the latches, thereby securing the plug in the aperture.
In a second embodiment of the invention, the pressurization of vessel is performed by a pick-and-place machine having a machine head for addressing the vessel. The machine head has an axial bore for communicating with the chamber of the vessel through the loading reservoir or aspiration port. The pick-and-place machine also includes a pressure source in fluid communication with the bore for pressurizing the chamber of the vessel through the bore. In this embodiment, the system also,preferably includes a disposable adapter for placing the bore in fluid communication with the chamber. The adapter is sized to be inserted into the seal aperture such that the adapter establishes a seal with the walls of the aperture. The adapter preferably includes a valve (e.g., a check valve) for preventing fluid from escaping from the vessel.
In a third embodiment of the invention, the device for increasing pressure in the chamber comprises an elastomeric plug, which is inserted into the seal aperture, and a needle, which is inserted through the plug for injecting fluid into the vessel. The needle is used to inject air or another suitable gas to increase pressure in the chamber. The reaction vessel may be pressurized in this manner by a human operator, or alternatively, the system may include an automated machine for filling and pressurizing the chamber. In the automated embodiment, the system includes a machine for inserting the needle through the plug, and the machine includes a pressure source for injecting fluid into the vessel through the needle.
In a fourth embodiment of the invention, the device for pressurizing the chamber comprises a platen for heat sealing a film or foil to the vessel. The foil is sealed to the portion of the loading structure defining the seal aperture.
Heat sealing the film or foil to the vessel in this manner seals the aperture and reduces the volume capacity of the vessel, thereby increasing pressure in the chamber. The reaction vessel may be heat sealed in this manner by a human operator, or alternatively, the system may include an automated machine, e.g. a press, for sealing the vessel.
The system of the present invention permits real-time monitoring and detection of analytes in the vessel with improved optical sensitivity. In the preferred embodiment, at least two of the side walls of the chamber are optically transmissive and angularly offset from each other, preferably by an angle of about 90xc2x0. The system further comprises optics for optically interrogating the sample contained in the chamber through the optically transmissive side walls. The optics include at least one light source for exciting the sample through a first one of the side walls, and at least one detector for detecting light emitted from the chamber through a second one of the side walls.
Optimum optical sensitivity may be attained by maximizing the optical sampling path length of both the light beams exciting the labeled analytes in the sample and the emitted light that is detected. The thin, wide reaction vessel of the present invention optimizes detection sensitivity by providing maximum optical path length per unit analyte volume. In particular, the vessel is preferably constructed such that the ratio of the width of the chamber to the thickness of the chamber is at least 4:1, and such that the chamber has a thickness of less than 3 mm. These parameters are presently preferred to provide a vessel having a relatively large average optical path length through the chamber, while still keeping the chamber sufficiently thin to allow for extremely rapid heating and cooling of the sample.
The system of the present invention may be configured as a small hand-held instrument, or alternatively, as a large instrument with multiple reaction sites for simultaneously processing hundreds of samples. In high throughput embodiments, the plates, heating and cooling elements, and optics are preferably disposed in a single housing to form an independently controllable, heat-exchanging module with. detection capability. The system includes a base instrument for receiving a plurality of such modules, and the base instrument includes processing electronics for independently controlling the operation of each module. Each module provides a reaction site for thermally processing a sample contained in a reaction vessel and for detecting one or more target analytes in the sample. The system may also include a computer for controlling the base instrument.